The cAMP-dependent protein kinase (PKA) regulates a wide array of cellular functions. In brain and heart PKA increases the activity of the L-type Ca2+ channel Cav1.2 in response to beta-adrenergic stimulation. Cav1.2 forms a complex with the beta2-adrenergic receptor, the trimeric GS protein, adenylyl cyclase, and PKA wherein highly localized signaling occurs [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Burette, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. PKA primarily phosphorylates Cav1.2 on serine 1928 of the central, pore-forming alpha11.2 subunit. Here we demonstrate that the A-kinase anchor protein 150 (AKAP150) is critical for PKA-mediated regulation of Cav1.2 in the brain. AKAP150 and MAP2B specifically co-immunoprecipitate with Cav1.2 from rat brain. Recombinant AKAP75, the bovine homologue to rat AKAP150, binds directly to three different sites of alpha11.2. MAP2B from rat brain also interacts with these same sites in pull-down assays. Gene disruption of AKAP150 in mice dramatically reduces co-immunoprecipitation of PKA with Cav1.2 and prevents phosphorylation of serine 1928 upon beta-adrenergic stimulation in vivo. These results demonstrate the physiological relevance of PKA anchoring by AKAPs in general and AKAP150 specifically in the regulation of Cav1.2 in vivo.
The cAMP-dependent protein kinase (PKA) controls a large number of cellular functions. One critical PKA substrate in the brain and heart is the L-type Ca(2+) channel Ca(v)1.2, the activity of which is upregulated by PKA. The main PKA phosphorylation site is serine 1928 in the central pore forming alpha(1)1.2 subunit of Ca(v)1.2. PKA is bound to Ca(v)1.2 within a macromolecular signaling complex consisting of the beta(2) adrenergic receptor, trimeric G(s) protein, and adenylyl cyclase for fast, localized, and hence specific signaling [Davare, M. A., Avdonin, V., Hall, D. D., Peden, E. M., Buret, A., Weinberg, R. J., Horne, M. C., Hoshi, T., and Hell, J. W. (2001) Science 293, 98-101]. Protein phosphatase 2A (PP2A) serves to effectively balance serine 1928 phosphorylation by PKA through its association with the Ca(v)1.2 complex [Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717]. We now show that native PP2A holoenzymes, as well as the catalytic subunit itself, bind to alpha(1)1.2 immediately downstream of serine 1928. Of those holoenzymes, only heterotrimeric PP2A containing B' and B' ' subunits copurify with alpha(1)1.2. Preventing the binding of PP2A by truncating alpha(1)1.2 28 residues downstream of serine 1928 hampers its dephosphorylation in intact cells. Our results demonstrate for the first time that a stable interaction of PP2A with Ca(v)1.2 is required for effective reversal of PKA-mediated channel phosphorylation. Accordingly, PKA as well as PP2A are constitutively associated with Ca(v)1.2 for its proper regulation by phosphorylation and dephosphorylation of serine 1928.
Antibiosis is one of the widespread strategies used by Trichoderma spp. against plant fungal pathogens, the mechanism of which, however, remains poorly understood. Peptaibols are a large family of antimicrobial peptides produced by Trichoderma spp. Our previous study showed that trichokonins, a type of peptaibol from Trichoderma pseudokoningii SMF2, exhibited antibiotic activities against plant fungal pathogens. In this study, we first demonstrated that trichokonin VI (TK VI) induced extensive apoptotic programmed cell death in plant fungal pathogens. For a deeper insight into the apoptotic mechanism involved in the action of TK VI, Fusarium oxysporum was used as a model. Cells of F. oxysporum treated with TK VI showed apoptotic hallmarks, such as exposure of phosphatidylserine, the appearance of reactive oxygen species and fragmentation of nuclear DNA. Moreover, TK VI-treated cells exhibited an accumulation of cytoplasmic vacuoles with loss of the mitochondrial transmembrane potential, and this process was independent of metacaspases. Therefore, TK VI induces metacaspase-independent apoptotic cell death in F. oxysporum. This represents what is believed to be the first report to reveal the antibiotic mechanism of peptaibols against plant fungal pathogens. INTRODUCTIONSome Trichoderma species, such as Trichoderma harzianum, Trichoderma virens and Trichoderma viride, are commercial biological control agents (BCAs) against plant fungal pathogens (Chet & Inbar, 1994;Howell, 2003;Benítez et al., 2004). Trichoderma spp. act as BCAs through mycoparasitism, antibiosis, substrate competition, and production of antibiotics and cell wall-degrading enzymes (CWDEs) to inhibit the growth of fungal pathogens, promote plant growth and induce resistance in plants (Yedidia et al., 1999;Viterbo et al., 2005). Antibiosis is a widespread strategy used for defence by Trichoderma spp. (Pyke & Dietz, 1966;Whitmore & Wallace, 2004). Trichoderma spp. produce a variety of antibiotics, such as gliovirin, gliotoxin, viridin, pyrones and peptaibols, against fungal phytopathogens (Howell, 2003;Harman et al., 2004). However, among the reported biocontrol mechanisms of Trichoderma, very few studies have been done on their antibiotic mechanism.Peptaibols are a large family of antibiotic peptides from soil fungi, including Trichoderma and related genera such as Emericelopsis and Gliocladium (Daniel & Filho, 2007). To date, 317 peptaibols have been reported, and among them more than 190 are synthesized by Trichoderma. Details are compiled in the Peptaibol Database (http://www.cryst.bbk. ac.uk/peptaibol) (Whitmore & Wallace, 2004). Peptaibols are characterized by the presence of an unusual amino acid, a-aminoisobutyric acid (Aib), a C-terminal-hydroxylated and N-terminal-acetylated amino acid, and they are able to form voltage-dependent ion channels in lipid bilayer membranes because of their linear and amphipathic nature (Chugh & Wallace, 2001). Most research on peptaibols has focused on their biosynthetic pathways, conformational properties and a...
Hydrotreatment of nonedible jatropha oils over PtPd/Al 2 O 3 catalyst and NiMoP/Al 2 O 3 catalysts was investigated under conditions of 330−390 °C, 3 MPa and 2 h −1 in a fixed-bed reactor. A significantly high yield range of about 82 wt % of liquid hydrocarbon products was achieved over all catalysts. Moreover, the liquid hydrocarbon products have low acid number, suitable density and viscosity, and quite high cetane index. The oil can be a high-performance additive for diesel oil. The oxygen removal pathway of jatropha oil over PtPd/Al 2 O 3 catalyst is primarily compiled through decarboxylation and/or decarbonylation, but over NiMoP/Al 2 O 3 catalysts, the oxygen removal pathways are executed primarily by hydro-deoxygenation. A long-term experiment was conducted over catalyst A (NiMoP/Al 2 O 3 ). Results show that catalyst A starts deactivation from reaction time of 120 h. The sulfide catalyst conversion to oxide catalyst is regarded as the main reason for deactivation. The deactivated catalyst can be reused after regeneration treatment.
a b s t r a c tTo investigate the role of microRNAs in the development of chemoresistance and related epithelialmesenchymal transition (EMT), we examined the effect of miR-489 in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). MiR-489 was significantly suppressed in MCF-7/ADM cells compared with chemosensitive parental control MCF-7/WT cells. Forced-expression of miR-489 reversed chemoresistance. Furthermore, Smad3 was identified as the target of miR-489 and is highly expressed in MCF-7/ADM cells. Forced expression of miR-489 both inhibited Smad3 expression and Smad3 related EMT properties. Finally, the interactions between Smad3, miR-489 and EMT were confirmed in chemoresistant tumor xenografts and clinical samples, indicating their potential implication for treatment of chemoresistance.
Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.
BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.ResultsHere, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of μ-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.ConclusionTaken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.
Polydatin and resveratrol, as major active components in Polygonum cuspidatum, have anti-inflammatory, antioxidant and antitumor functions. However, the effect and mechanism of polydatin and resveratrol on enterovirus 71 (EV71) have not been reported. In this study, resveratrol revealed strong antiviral activity on EV71, while polydatin had weak effect. Neither polydatin nor resveratrol exhibited influence on viral attachment. Resveratrol could effectively inhibit the synthesis of EV71/VP1 and the phosphorylation of IKKα, IKKβ, IKKγ, IKBα, NF-κB p50 and NF-κB p65, respectively. Meanwhile, the remarkably increased secretion of IL-6 and TNF-α in EV71-infected rhabdosarcoma (RD) cells could be blocked by resveratrol. These results demonstrated that resveratrol inhibited EV71 replication and cytokine secretion in EV71-infected RD cells through blocking IKKs/NF-κB signaling pathway. Thus, resveratrol may have potent antiviral effect on EV71 infection.
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